SUPPLEMENTARY MATERIAL for the following paper:
Targeted modification of storage protein content resulting in improved amino acid composition of barley grain
Md. S. I. Sikdar1,2, S. Bowra3, D. Schmidt1,4, G. Dionisio1, P. B. Holm1 and E. Vincze1
1Aarhus University, Science and Technology, Dept. of Molecular Biology and Genetics, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark.
2Department of Agronomy, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200, Bangladesh.
3 Verzyme (UK) Ltd., PlasGogerddan, Aberystwyth, Wales SY23 3EB, United Kingdom
4Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, SP, CEP 13418-900, Brazil
Correspong author Eva Vincze,
Email: ,
Telephone number: +45 8715 8242
Table S1SDS-PAGE protein band identification by nanoUPLC/Q-TOF MS/MS. SDS-PAGE gel bands were numbered according to Fig. S3; isolated from the gel; digested by chymotrypsin; and identified by nanoUPLC/Q-TOF MS/MS. The PLGS score is an indication of peptide/protein abundance judged by their spectral counting
Band# / Accession / Entry / Description / mW (Da) / pI (pH) / PLGS Score / Peptides / Coverage (%)
1 / I6SW34 / I6SW34_HORVD / D-hordein / 79303 / 7.9585 / 4516 / 2 / 5.0
2 / I6TEV8 / I6TEV8_HORVD / C-hordein / 35291 / 9.4951 / 54033 / 16 / 28.4
3 / I6TEV8 / I6TEV8_HORVD / C-hordein / 35291 / 9.4951 / 11301 / 3 / 11.6
4 / Q41210 / Q41210_HORVU / C-hordein / 36485 / 7.7139 / 85778 / 9 / 25.1
Q40053 / Q40053_HORVU / Hor1 17 C-hordein / 30377 / 7.645 / 43459 / 6 / 8.4
5 / Q41210 / Q41210_HORVU / C-hordein / 36485 / 7.7139 / 172565 / 34 / 28.1
Q40053 / Q40053_HORVU / Hor1 17 C hordein / 30377 / 7.645 / 106090 / 43 / 25.7
6 / Q3YAF9 / Q3YAF9_HORVD / B-hordein / 34465 / 8.67 / 2341109 / 4 / 14
P06471 / HOR3_HORVU / B3-hordein / 30195 / 7.6 / 2712945 / 2 / 11.4
7 / I6TEV5 / I6TEV5_HORVU / B3-hordein / 35440 / 7.5146 / 54185 / 10 / 15.8
I6SJ26 / I6SJ26_HORVU / B3-hordein / 35402 / 8.0874 / 21483 / 11 / 18.4
I6SJ17 / I6SJ17_HORVD / γ1-hordein / 34714 / 7.9541 / 13871 / 1 / 3.6
8 / I6SJ22 / I6SJ22_HORVU / B1-hordein / 32776 / 8.2969 / 211676 / 35 / 18.5
I6TMV6 / I6TMV6_HORVU / γ1-hordein / 34640 / 7.9541 / 129089 / 38 / 39.1
I6SJ17 / I6SJ17_HORVD / γ1-hordein / 34714 / 7.9541 / 14056 / 23 / 17.7
Q3YAF9 / Q3YAF9_HORVD / B-hordein / 34442 / 8.6763 / 70171 / 15 / 7.7
I6QP72 / I6QP72_HORVD / B-hordein / 33481 / 7.0562 / 64906 / 24 / 7.9
Q2XQF1 / Q2XQF1_HORVD / B-hordein / 30136 / 7.8574 / 22717 / 6 / 14.2
9 / I6SJ22 / I6SJ22_HORVU / B1-hordein / 32776 / 8.2969 / 32382 / 6 / 16.8
I6TMV6 / I6TMV6_HORVU / γ1-ordein / 34640 / 7.9541 / 30361 / 22 / 30.5
I6TEV2 / I6TEV2_HORVU / γ3-hordein / 33763 / 6.7046 / 7668 / 6 / 9.2
10 / B5TWK6 / B5TWK6_HORVU / rRNA N glycosidase / 29532 / 10.103 / 64122 / 5 / 13
I6SJ17 / I6SJ17_HORVD / γ1-hordein / 34714 / 7.9541 / 2615 / 2 / 4.3
11 / F2CWL7 / F2CWL7_HORVD / Oleosin / 18496 / 10.021 / 36352 / 4 / 20.1
F2EI31 / F2EI31_HORVD / Alpha-amylase/trypsin inhibitor BDAI-1 / 16605 / 5.1899 / 22841 / 9 / 42.2
F2CRF2 / F2CRF2_HORVD / Oleosin / 16091 / 9.5889 / 18523 / 2 / 7.5
12 / F2EI31 / F2EI31_HORVD / Alpha-amylase/trypsin inhibitor BDAI-1 / 16605 / 5.1899 / 255814 / 41 / 56.5
E7BB45 / E7BB45_HORVD / Barley trypsin inhibitor CMc / 11241 / 7.4399 / 30296 / 16 / 76
Q5IUH9 / Q5IUH9_HORVD / Hordoindoline b1 / 16110 / 8.2969 / 23826 / 11 / 14.3
13 / F2EI31 / F2EI31_HORVD / Alpha-amylase/trypsin inhibitor BDAI-1 / 16605 / 5.1899 / 284504 / 43 / 52.6
F2ED95 / F2ED95_HORVD / Non-specific lipid transfer protein / 12323 / 8.3057 / 131753 / 20 / 45.3
E7BB45 / E7BB45_HORVD / Barley trypsin inhibitor CMc / 11241 / 7.4399 / 31333 / 10 / 76
Table S2 Statistical analysis of selected amino acid from RNAi lines compared to non-transgenic parental line using the independent samples t-test(Schmider et al. 2010)
Selected amino acidsGenoptype / Proline / Glutamine / Lysine / Threonine / Methionine / Leucine
Parental line
Mean / 16.16 / 24.53 / 3.21 / 3.66 / 0.78 / 7.12
skewness / 1.73 / 0.14 / 1.77 / 1.58 / 1.52 / 3.60
kurtosis / 0.32 / 0.27 / 0.41 / 0.07 / 0.39 / 1.78
t- test of C-hordein RNAi line: Mean vs. Non Transgenic Parental line
Mean / 13.79 / 23.03 / 3.54 / 4.06 / 1.08 / 7.86
skewness / 0,23 / 1.05 / 0.53 / 1.99 / 0.75 / 0.85
kurtosis / 0.43 / 0.34 / 0.35 / 0.01 / 0.52 / 0.42
t-value / 3.51 / 2.04 / 2.27 / 4.89 / 4.68 / 4.68
p value / 0.015 / 0.045 / 0.033 / 0.001 / 0.002 / 0.002
t- test of ω-gliadin RNAi line: Mean vs. Non Transgenic Parental line
Mean / 13.67 / 22.50 / 3.58 / 4.05 / 0.94 / 7.49
skewness / 1.06 / 1.05 / 0.90 / 0.31 / 0.87 / 1,78
kurtosis / 0.16 / 0.34 / 0.81 / 0.72 / 0.76 / 0.39
t-value / 4.36 / 3.48 / 2.02 / 4.13 / 2.08 / 3.19
p value / 0.003 / 0.008 / 0.049 / 0.004 / 0.018 / 0.012
Fig S1Characterisation of the transgenic lines by PCR.Panel A: 1: DNA marker (1 kb ladder); 2: Golden Promise control; 20, 22, 23, 27, 32, 33, 48, 51, 52, 2,3, 9, 14 C-hordein RNAi transgenic lines; Panel B: DNA marker (1 kb ladder; Golden Promise control; 116-130 ω-gliadin RNAi transgenic lines.
Fig.S2Characterisation of the transgenic lines bySouthern blot hybridisation. Panel A: M: DNA marker (1 kb ladder); GP: Golden Promise control; 4, 5, 6, 8, 11, 19, 20, 27, 32, 48, 52 C-hordein RNAi transgenic lines; Panel B: M: DNA marker (1 kb ladder; GP: Golden promise control; 100, 113, 114, 118,119, 120, 122, 130, 135, 143 ω-gliadin RNAi transgenic lines.
Fig.S3Hordein extraction and separationfrom mature barley grains of parental line cv. Golden Promise for MS/MS analysis.The alcohol soluble proteins were isolated and separated by SDS-PAGE. The bands were numbered, isolated from the gel and the proteins were identified with quadrupole-time-of-flight (qTOF) mass spectrometry. The list of the protein could be found in Table S1.
Fig. S4 The hordein patterns of C-hordein RNAi double insertion line number 22(T1grain) andthe T2 grains (line 31 and 33).